To understand the role of the Slack gene in neurons and other excitable cells, it is essential to characterize the heteromeric channels resulting from the co-expression of Slack and Slo subunits. In this project we will identify the subunit stoichiometries that result in functional channels and characterize these channels in regard to their voltage and Ca sensitivity and conductance properties. We will also test for the functional properties of "core/tail" constructs of Slack subunits created in Project 2. Further, we will establish systems for expression and purification of Slack and Slo protein; with purified, reconstituted channel protein, we will address the three-dimensional structure of the Maxi-K channel complex. Heretofore the only direct structural information about the six-transmembrane-segment family of ion channel proteins consists of crystal structures of cytoplasmic domains and low-resolution, projection EM images of negatively-stained channel proteins. We will obtain three-dimensional structural information at moderate resolution of Maxi-K channels using the combination of cryo-electronmicroscopy and novel single-particle reconstruction techniques.